This project is designed to define the morphologic, molecular, and metabolic characteristics of breast ducts and ductal epithelial cells at normal risk and increased risk for breast cancer among Caucasian, Hispanic and African American women. This information is needed to define the early changes in the carcinogenic pathway for breast cancer, to develop an improved classification and molecular signature of preneoplastic breast tissue for risk assessment, to identify new targets and to facilitate selection and monitoring of women for breast cancer prevention, and to define the molecular basis for disparities in the development and presentation of breast cancer. This project includes the following clinical and laboratory studies: a.) Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and Gene Expression Profile (DN Danforth, PI). b.) Protocol 02-C-0144, Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer (DN Danforth, PI). c.) Comprehensive literature review of molecular changes in normal breast tissue at risk for breast cancer to define molecular changes in early breast carcinogenesis and guide the molecular characterization of breast epithelial cells collected from women at risk for breast cancer. d.) Comprehensive literature review of molecular changes contributing to disparities in development, presentation and outcomes of breast cancer between Caucasian, Hispanic, and African American women. e.) Regulation of breast epithelial proliferation by the endogenous risk factors estradiol and IGF-1. A comprehensive review has recently been published proposing for the first time a model describing the relationship of biological and nonbiological factors to the initiation and development of the major disparities in breast cancer between African American and Caucasian women (Danforth, DN Breast Cancer Research, 15:208, 2013). This model identifies multiple molecular differences in breast cancer between African American and Caucasian women, and these differences are the major drivers of the disparities in age of onset, more advanced stage, more aggressive histology, and worse survival in African vs. Caucasian women. Multiple socioeconomic, reproductive and health care factors influence the outcome of the disparities through their influence on the breast cancer molecular characteristics. This model also emphasizes the need for defining the molecular characteristics of early carcinogenesis in these ethnic groups. Protocol 02-C-0077 characterizes by ductal lavage and ductal endoscopy the breast ducts and ductal epithelium of African American women at normal risk and increased risk for breast cancer. One hundred seventeen women have been studied, 53 high risk subjects and 64 subjects at normal risk. The ductal endoscopic architectural characteristics of breasts have been defined and correlated with the presence of epithelial cell atypia. A significantly improved method of ductal epithelial cell sampling has been developed which provides multiple samples of pure (90%) ductal epithelial cells with high cellularity. Extraction methods have been developed for samples which provide significant quantities of high grade DNA and RNA suitable for molecular profiling. Cytologic evaluation and quantitative and qualitative cellular assessment is performed for each aliquot. Molecular studies to define numerical and structural chromosome abnormalities and gene and microRNA expression of normal at-risk breast epithelial cells from Caucasian, Hispanic and African American women are in progress. Chromosomal numerical and structural changes will be studied on Genomic DNA using a SNP-CGH microarray (SurePrint G3, Agilent technologies). Ploidy, loss of heterozygosity, hemizygous and homozygous deletions, and copy number aberrations will be determined. Allelic losses or gains willbe validated with gene-specific TaqMan Copy Number Assays (Applied Biosystems). Quantitative assessment of telomere length will be determined by qRT-PCR according to the methods of Das et al. The identification of telomeric shortening may indicate cells at significant risk for chromosomal instability and progression in the carcinogenic pathways. Gene expression profiling is performed on total RNA which is amplified (Low Input Quick Amp Kit, Agilent) and hybridized to Agilent Human Gene Expression 4 x 44 microarray. Gene expression will be analyzed by unsupervised hierarchical clustering to identify innate expression differences between the two ethnic groups, and by supervised analysis to identify potentially ethnic-specific gene expression patterns and signaling pathways. Breast ductal fluid and ductal epithelial cells are being studied for the presence of miRNA, noncoding transcripts which bind to mRNA and result in gene silencing. miRNA has been identified in exosomes of breast ductal fluid, suggesting an important mechanism for the expansion of the cancerized field within the breast and enhancement of progression through the carcinogenic pathway. The effects of two prominent endogenous mitogenic risk factors for breast cancer, estradiol (E2) and insulin-like growth factor-1 (IGF-1) on normal and high risk breast epithelial cells are being studied to further define regulation of proliferation in early carcinogenesis. IGF-1 stimulated growth of all breast epithelial cells without modulation of apoptosis. This was accompanied by the rapid stimulation of phosphorylation of IGF-1R and IRS-1, and by downregulation of IRS-1 at the posttranscriptional level and of IRS-2 at the transcriptional level. E2 did not stimulate proliferation or cell cycle progression, however, IGF-1 acted synergistically with E2 to stimulate growth in a high risk breast epithelial cell line (MCF12A) but not in any normal risk cell lines (MCF10A, AG11132, AG11134), suggesting that the transition to estradiol responsiveness and synergism with IGF-1 may occur at or beyond the level of hyperplasia in the carcinogenic pathway. Further, these findings indicate that IGF-1 is the dominant mitogen in early breast carcinogenesis, and estrogen responsiveness of normal breast epithelial cells and modulation of IGF-1 signaling occur later in the carcinogenic pathway. Recent studies have identified important abnormalities in G1/S phase proteins and related tumor suppressor genes in African American breast cancer which may contribute to the earlier age of onset and more aggressive histology in these women, two major disparities in breast cancer. To further define these changes the structural chromosomal and gene expression changes of breast cancer cell lines developed from breast cancer in African Americans will be compared to those of Caucasian women using a SNP-CGH microarray (SurePrint G3, Agilent technologies) and gene expression profiling (Agilent Human Gene Expression 4 x 44 microarray). Analysis of proteins involved in cell cycle progression, mitotic checkpoint pathway and spindle assembly checkpoint pathways will be emphasized. The identification of specific abnormalities between Caucasian and African American breast cancer will also provide important correlative information for analysis of normal and high risk breast epithelium, and facilitate characterization of carcinogenic changes in at-risk breast epithelium.